Currently, in the environment, there are various types of artificial chemicals, for example, various carcinogenic substances, endocrine disturbing substances and so forth originated from pharmaceutical preparations, agricultural chemicals, detergents, food additives, antiseptics and so forth, that have not existed in the natural environment. Among the natural substances, there are substances on whose toxicity must be taken care because:    1) There is the possibility that deleterious components can exist in natural materials;    2) There is the possibility that in particular when it is formulated in the form of an extract, its biological toxicity is enhanced; and    3) There is the possibility that various components synergistically act to become toxic.
Genetic information is encoded by base pairs of adenine (A), thymine (T), guanine (G) and cytosine (C). The DNA information is injured at a certain frequency due to UV light irradiation, DNA replication error or exposure to various carcinogenic substances or active oxygen species, and ultimately loss, recombination, mutation and so forth of the gene information occurs, thus causing death of cells or the individual or on the contrary triggering naissance of new biological species. The mechanism in which the natural and synthetic chemicals exerts toxicity to animal including humans can be partly explained by such DNA injury.
From this aspect, focusing attention on the oxidation of DNA, 2′-deoxyguanosine (dG) and 2′-deoxyadenosine (dA), which are DNAs (mononucleosides) having purine nuclei bind with hydroxy radicals (.OH), which are active oxygen species, to be oxidized into 8-hydroxy-2′-deoxyguanosine (8OHdG) and 2-hydroxy-2′-deoxyadenosine(2OHdA), respectively. And then the DNA base pairs that should inherently be G:C and A:T are converted to T:A and G:C, respectively. The frequency of occurrence of mistranslation of gene information on the level of DNA is considered to be important information relative to states of various diseases and paid much attention in recent medicinal fields.
Conventionally, biologically acceptable safe concentration of chemicals has been quantified in consideration of    1) Lethal dose when administered to test animals;    2) Amount in which apparent organ disorder such as carcinogenesis or neuropathy occurs when administered to test animals;    3) Amount that influences the fecundity and so forth and by multiplying such minimum effective amount by a safety factor of 100 to 1,000 times.
However, because there are extremely many types of chemicals which are evaluation targets; i.e., as many as at least about 100,000 types, because in the case where biological toxicity is quantified by using mouse or rat, it takes about 2 months for establishing a first generation and a double or more days are necessary for evaluating influence over generations, and because a huge cost is necessary for maintaining the evaluation system and for some other reasons, it has conventionally been practically impossible to evaluate the biological toxicity of all the substances by the conventional method due to restrictions of time and costs.
Many foods currently on the market such as health-care foods and functional foods are in the main those extracted from natural materials or those to which a specific substance is added in the range of safe acceptable amount. However, no objective evaluation system on usefulness (effectiveness) of the foods and the like has been established.
On the other hand, as for a method for measuring 8-hydroxy-2′-deoxyguanosine (8OHdG), which is one of oxidized DNA substances as described above, in biological samples such as leukocytes, parenchyma organ or cell suspension, measurement has been generally performed by extracting nuclear DNA from these cell components and subjecting it to various enzyme treatments, passing it through HPLC (high performance liquid chromatography), and flowing the eluate to an electrochemical detector to measure it in the form of mononucleoside. This method can evaluate oxidation injury of nuclear DNA per se at measurement sensitivity in the order of 10 pg/ml. However, the method involves steps of acid or alkali treatment, enzyme reaction and so forth so that the operation of it is troublesome and secondary DNA oxidation is inevitable due to these treatments so that the method can be practiced only in limited installment.
Likewise, in the case where 8OHdG is measured in urine that contains it in a large amount, a method is used in which after removal of proteins, it is passed through HPLC and an electrochemical detector. However, since urine per se is a biological sample that is very susceptible to oxidation, it has been the most difficult obstacle course how to remove proteins under conditions where oxidation is prevented.
A method for quantifying 8OHdG that is increasingly used recently is an enzyme antibody method (EIA method) by use of monoclonal antibody. Unlike the method by use of HPLC and an electrochemical detector, this method is characterized in that it can detect even when the sample is not in the form of mononucleoside. However, it has defects that it undergoes cross-reaction with other substance than 8OHdG and that it has low detection sensitivity in the order of 1 ng/ml. Accordingly, there have been many problems to be solved in order to generalize it.
On the other hand, it is relatively easy to detect mononucleoside per se in a biological sample since the absolute amount of it in nuclear DNA is relatively large; i.e., about 10,000 to 100,000 times that of oxidized products such as 8OHdG, and due to the characteristics of its chemical structure, UV light absorption measurement apparatus is effectively used in a general detection method. However, pretreatment of analyte to be measured is the same as in the measurement of 8OHdG by use of an electrochemical detector and hence basically the same problems are involved.
It is possible that the artificial chemicals and foods as described above are daily taken by, administered to or brought into contact with humans and therefore it is an urgent necessity to confirm biological toxicity and usefulness, in particular toxicity. However, the present situation is as described above and there are problems in that a long time and high costs are necessary. Accordingly, development of a method for evaluating biological toxicity of artificial chemicals or harmfulness and usefulness of foods in a simplified manner and at low costs has been demanded.
The present invention has been made in response to such a demand and an object of the present invention is to provide a method for evaluating biological harmfulness or usefulness of huge kinds of natural substances and artificial chemicals and foods by in a rational and simplified manner.
Also, an object of the present invention is to provide a measurement apparatus that can accurately quantify in a simplified manner with good reproducibility the amounts of substances that serve as indices in a biological evaluation method, more broadly, oxidized DNA substances, in particular 8-hydroxy-2′-deoxyguanosine (8OHdG), 2-hydroxy-2′-deoxyadenosine (2OHdA) and the like together with the amount of nuclear DNA in the biological sample.
Furthermore, an object of the present invention is to provide an antioxidative preservative solution being able to prevent oxidation injury of DNA in the biological sample in the aforementioned measurement apparatus.